The Development Of Chloroplast SSR(SNP) Markers For Miscanthus Species

Miscanthus Andersson (Poaceae) has attracted considerable attention as one of the most promising non-food energy crops because of its high biomass yield and relatively low input. China is the distribution center of Miscanthus species but so far little is known about their genetic diversity. Chloroplast simple sequence repeat (cpSSR) marker is one of powerful molecular tools for studying Miscanthus population structure, diversity and phylogeography. In this study, a set of novel chloroplast microsatellite markers (cpSSRs) were developed for Miscanthus and their ability to cross species amplification was evaluated. The main results are as follows:(1) A total of36cpSSR loci were identified from a complete chloroplast genome sequence of Saccharum officinarum, a species closely related to Miscanthus and twenty-eight locus-specific PCR primer pairs were designed from the sequences flanking SSR loci. But only17of the28primers screened amplified the target SSR loci successfully from Miscanthus chloroplast DNA. Of these sixteen loci were mononucleotide microsatellites including9pure repeats,3interrupted repeats and4compound repeats. Furthermore,(A)n and (T)n were the major repeats in mononucleotides.(2) Cross-species amplification of novel cpSSR loci was tested on64individuals from8Miscanthus taxa, including20accessions of M. sinensis,20accessions of M. sacchariflorus,10accessions of M. floridulus,10accessions of M. lutarioriparius, and1accession each of M. x giganteus (an allotriploid originating from Japan), M. nepalensis, M. nudipes, and M. paniculatus. All17primers were then tested, among which16cpSSR loci were found to be polymorphic. The number of alleles per polymorphic locus ranged from2to7, with an average3.94alleles. The unbiased haploid diversity index (h) per polymorphic locus was from0.062to0.715.(3) Several single nucleotide polymorphism (SNP) loci that are species-specific were identified from the flanking sequences of some cpSSR loci, they can be used for studying the relationship among Miscanthus species.